Neutrophils are a normal constituent of the female reproductive tract and their numbers increase in the late secretory phase of the menstrual cycle prior to menses. Several cytokines are produced in female reproductive tract tissue. In particular granulocyte-macrophage colony-stimulating factor (GM-CSF), a potent activator of neutrophils, is secreted in high concentrations by female reproductive tract epithelia. We previously observed that GM-CSF synergizes strongly with interleukin-8 (IL-8) in enhancing chemotaxis of neutrophils. Thus we investigated whether pretreatment of neutrophils with GM-CSF would prime subsequent chemotaxis to IL-8 in the absence of GM-CSF. Surprisingly, a 3-hr pulse of GM-CSF severely diminished chemotaxis to IL-8, whereas N-formyl-methyl-leucyl-phenylalanine (fMLP)-mediated chemotaxis was retained. Conversely, when cells were incubated without GM-CSF they retained IL-8-mediated migration but lost fMLP chemotaxis. These changes in chemotaxis did not correlate with expression of CXCR1, CXCR2 or formyl peptide receptor. However, IL-8-mediated phosphorylation of p44/42 mitogen-activated protein kinase was greatly reduced in neutrophils that no longer migrated to IL-8, and was diminished in cells that no longer migrated to fMLP. Oestradiol, which is reported by some to exert an anti-inflammatory effect on neutrophils, did not change the effects of GM-CSF. These data suggest that neutrophil function may be altered by cytokines such as GM-CSF through modulation of signalling and independently of surface receptor expression.